Human Epidermal Growth Factor Receptor Her2, also known as Neu, ErbB-2, or p185, is a member of the epidermal growth factor receptor (EGFR/ErbB) family and encoded by the ERBB2 gene. Herein, the terms “HER2” or “Her2” and “Her2/neu” are used interchangeably. As other members of ErbB family, Her2 is a membrane-bound receptor tyrosine kinases composed of extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with downstream signaling molecules. Unlike the other family members, HER2 is considered to be an orphan receptor as it has no known ligand. HER2 can heterodimerise with other ErbB family receptors and is considered to be their preferred dimerisation partner. Dimerisation results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to proliferation and inhibition of apoptosis.
Amplification of the ERBB2 gene occurs in 20-30% of human breast and ovarian cancers and is linked to a more aggressive disease course and worse prognosis (Bange, J., Zwick E. & Ullrich A., 2001, Nature Medicine, 7: 548-552; Slamon, D. J., Clark, G. M., Wong, S. G. et al., 1987′ Science, 235:177-182; Slamon, D. J., Godolphin, W., Jones, L. A. et al., 1989, Science, 244:707-712; Berchuck, A., Kamel, A., Whitaker. R., et al., 1990, Cancer Research 50:4087-4091). In ERBB2+ tumor cells, the receptor can function on its own and/or it needs to heterodimerize with another ErbB member to transduce a deregulated proliferative signal responsible for the neoplastic behavior of the cells.
In recent years HER2 has evolved as an important target for therapy of breast cancer in particular by monoclonal antibody therapy, e.g. Herceptin (trastuzumab) a humanized monoclonal antibody against this surface target has been approved by FDA in 1998. Herceptin has a significant impact on survival rates of HER2 positive breast cancer patients (Tan, A. R. & Swain, S. M., 2002, Seminars in Oncology, 30: 54-64). Although active against HER2 homodimers, trastuzumab is not effective against ligand-induced HER2 heterodimers (Agus, D. B., Akita, R. W, Fox, W. D., et al., 2002, Cancer Cell, 2:127-137; Cho, H. S., Mason, K., Ramyar, K. X., et al., 2003, Nature, 421:756-60). In addition, cancers usually develop resistance to trastuzumab (Cho, H. S., Mason, K., Ramyar, K. X., et al., 2003, Nature, 421:756-760). While trastuzumab is efficient for the treatment of late stage metastatic cancers, it is not clear if it is effective in earlier stage cancers (Editorial, 2005, Lancet, 366:1673).
In Jun. 2012, the FDA approved yet another monoclonal antibody pertuzumab (U.S. Pat. Nos. 7,449,184; 7,981,418) for treatment of HER2/neu positive metastatic breast cancer in combination with Herceptin® (trastuzumab) and docetaxel chemotherapy for patients who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease (Genentech press release on 8 Jun., 2012, available on the World Wide Web at: gene.com/gene/news/press-releases/display.do?method=detail&id=14007). This approach allowed extending patient's cancer progression-free period for median 6.1 months.
As an alternative or complementation to HER2/neu-positive cancers treatment with therapeutic antibodies, different cancer vaccines are currently under testing. This approach provides the most dramatic shift in cancer treatment, as patient's own immune system could be trained to recognise and delete HER2-positive cancer cells. Different vaccine designs (simple peptides, DNA encoding HER2 regions, HER2 protein fragments and whole-cell vaccines) have been tested in human clinical trials that have shown that significant levels of durable humoral or T-cell HER2 immunity can be generated with active immunization (Ladjemi, M. Z., Jacot, W., Charde's, T. et al., 2010, Cancer Immunol. Immunother., 59:1295-1312). The most advanced clinical studies are with peptide-based vaccines, especially the one based on E75 peptide (Disis, M. L., & K Schiffman, K., 2001, Semin Oncol., 28:12-20; Murray, J. L., Gillogly, M. E., Przepiorka, D. et al., 2002, Clin Cancer Res., 8:3407-3418; Peoples, G. E., Gurney, J. M., Hueman, M. T., et al., 2005, J Clin Oncol 23:7536-7545; Ross, J. S., Slodkowska, E. A., Symmans, W. F., et al., 2009, The Oncologist, 14:320-368; U.S. Pat. No. 8,222,214; for review see Ladjemi, M Z., Jacot, W., Chardes, T. et al., 2010, Cancer Immunol. Immunother., 59:1295-1312). This vaccination can break tolerance against endogenous HER2 receptor. The key component of the vaccine is peptide E75, a peptide of 9-amino acid residues (U.S. Pat. No. 8,222,214; Mittendorf, E. A., Clifton, G. T., Holmes, J. P. et al., 2012, Cancer, 118:2594-602). Cancer vaccines do not usually cause toxicities typically associated with the use of therapeutic antibodies or chemotherapeutics (e.g. Peoples, G. E., Gurney, J. M., Hueman, M. T., et at., 2005, J. Clin. Oncol., 23:7536-7545; Ross, J. S., Slodkowska, E. A., Symmans, W. F., et al., 2009, The Oncologist, 14:320-368; Dabney, R. S., Hale, D. F., Vreeland, T. J., et al., 2012, J. Clin. Oncol, 30 (ASCO suppl; abstr 2529); Hamilton, E., Blackwell, K., Hobeika, A. C., et al., 2012, J. Transl. Med., 10:28).
Also, no significant toxic autoimmunity directed against normal tissues has been encountered with vaccines (Bernhard, H., Salazar, L., Schiffman, K. et al., 2002, Endocr Re/at Cancer 9:33-44; Ladjemi, M. Z., Jacot, W., Charde's, T. et al., 2010, Cancer Immunol. Immunother., 59:1295-1312). Most of these vaccines focus on T-cell immunity and consist of peptides or mixes of a small number of epitopes, necessitating patient cohorts to be HLA-matched at enrollment, and consequently exhibit a narrow immune response (Ladjemi, M. Z., Jacot, W., Charde's, T. et al., 2010, Cancer Immunol. Immunother., 59:1295-1312).
In addition to a narrow spectrum of target sites for peptide vaccines, use of peptide-based vaccines might suffer from difficulties in identification of the most promising peptides. Peptide vaccines cause immune response limited to one or few peptides in the composition. A promising alternative are DNA vaccines that are easy to manufacture but, while they are superior to peptides in inducing CD8+T-cell responses (Chaise, C., Buchan, S. L., Rice, J. et al., 2008, Blood, 112:2956-2964; Rolla, S., Marchini, C., Malinarich, S. et al., 2008, Human Gene Therapy, 19:229-239; U.S. Pat. No. 8,207,141), they are typically less effective than protein vaccines in inducing antibody responses.
Clinical trials with HER2 protein-based vaccines were also carried out. In one case, the HER2 ICD (intra-cellular domain, aa 676-1255) was used as an adjuvant vaccine (Disis, M.L., Schiffman, K., Guthrie, K. et al., 2004, J. Clin. Oncol., 22:1916-1925). It was shown that the vaccine was well tolerated and the patients treated with the highest dose more rapidly developed immunity. No therapeutic effect was reported. Phase I clinical trials with dHER2 consisting of the complete extracellular domain (ECD) and a portion of the intracellular domain (ICD) in combination with a complex mix of adjuvants and the tyrosine kinase inhibitor Lapatinib showed no (cardio) toxic effect, but also showed weak T-cell responses and 55 days median time to progression (Hamilton, E., Blackwell, K., Hobeika, A. C. et al., 2012, J. Trans. Med., 10:28, available on the World Wide Web at: translational-medicine .com/content/10/1/28. Use of a portion of a HER2 ECD domain (aa 1-146) complexed with cholesteryl pullulan nanogels (CHP-HER2) was well tolerated by patients and induced T-cell responses to the truncated HER2 protein (Kitano, S., Kageyama, S., Nagata, Y. et al., 2006, Clin. Cancer. Res., 12:7397-7405). However, second trials with the same antigen showed that induced Abs (antibodies) did not recognize the HER2 antigen expressed in its native form at the surface of cancer cells (Kageyama, S., Kitano, S., Hirayama, M. et al., 2008, Cancer Sci., 99:601-6070). Combination of CHP-HER2 with yet another tumor-specific antigen, NY-ESO-1, did not lead to the improved immune responses. On contrary, there were weaker antibody responses to HER2 in the combination vaccine compared to CHP-HER2 alone (Aoki, M., Ueda, S., Nishikawa, H. et al., 2009, Vaccine, 27:6854-6861).
In summary, existing vaccines under development suffer from several limitations (Ladjemi, M. Z., Jacot, W., Charde's, T. et al., 2010, Cancer Immunol. Immunother., 59:1295-1312): whole tumor cell vaccines must be made individually, the (an) immune response difficult to monitor and there is a risk of auto-immunity induction in the presence of adjuvant; DNA vaccines carry a risk of potentially promoting the malignancy due to DNA integration into the cell genome; peptide vaccines generate immune response limited to one or few epitopes, can be degraded in the absence of adjuvant, have restricted HLA population; HER2 protein-based vaccines up to now have not shown significant therapeutic effect.
Esserman et al., Cancer Immunol. Immunother (1999) 47: 337-342 relates to vaccination of neu transgenic mice with the extracellular domain (ECD) of HER2. The authors report that immunization with the Neu ECD delayed the onset of tumor growth. However, the authors also report that once tumors began to grow, they appeared to do so at the same rate as those in the control immunized mice, i.e. without the Neu ECD antigen.
Schwaninger et al., Cancer Immunol. Immunother (2004) 53: 1005-1017 describe virosomes as a carrier system for Her2/neu cancer vaccines. Mice vaccinated with the extracellular domain (ECD) of the Her2/neu protein bound to virosomes generated a humoral and cytotoxic immune response. However, once tumors were formed in their mouse model, vaccination had no influence on tumor progression, i.e. had no therapeutic effect.
Therefore, it is an object of this invention to provide a protein conjugate suitable as a cancer vaccine for HER2/neu positive cancer. It is another object to provide a protein antigen and a protein conjugate that has a therapeutic effect on HER2 positive cancer. It is another object to provide a protein conjugate or cancer vaccine capable to eliciting a long-term T-cell dependent immune response. It is another object to provide a method of treating HER2/neu positive cancer using a vaccine that is capable of inducing strong protective humoral and cellular immune responses. It is another object of this invention to provide a formulation of said vaccine.